Therapy for injured muscles

ABSTRACT

Methods for treating an injured muscle by local administration of a neurotoxin, such as a botulinum toxin, to promote healing and/or to reduce the pain associated with an injured muscle.

BACKGROUND

[0001] The present invention relates to methods for treating muscleinjuries. In particular, the present invention relates to a method fortreating an injured muscle by administration of a neurotoxin to theinjured muscle.

[0002] Injuries to muscles include acute injuries to skeletal musclessuch as contusions (bruises), lacerations, ischemia, strains, andcomplete ruptures. These injuries may cause tremendous pain and canincapacitate the affected person, preventing them from being able to goto work or even to participate in normal daily activities. Of the acuteinjuries to skeletal muscles, strain (also known as stretch-inducedinjuries) is most common. For example, strains can account for up to 30%of all injuries treated by occupational or sports medicineprofessionals. Garrett et al. Am J Sports Med, 24(6):S2-S8, 1996.

[0003] A muscle strain injury is characterized by a disruption of amuscle-tendon unit. The disruption of the muscle-tendon unit may occuranywhere on the muscle. This type of injury most commonly occurs nearthe myotendinous junction (MTJ) of the superficial muscles workingacross two joints, such as the rectus femoris, semitendinousus andgastroenemius muscles.

[0004] Muscle strain may result from an eccentric exercise, or uncommonuse of the muscle. For example, eccentric contractions employ feweractive motor units to generate higher forces. In such case, theover-extended muscle units experience excessive tension duringlengthening. The excessive tension may cause microscopic damages to thecontractile element of the muscle, centering on what appears to berandom disruptions of the Z-lines. When the muscle is damaged, theaffected person may experience a delayed onset muscle soreness,characterized by pain, weakness and a limited range of motion. The painis most intense for about 1 to 2 days after the muscle injury and theweakness and limited range of motion can last for a week or more. If aminor strain of the skeletal muscles is treated inappropriately, a moreserious injury can occur.

[0005] There are three classifications of muscle strains, based on theseverity of the injury and the nature of the hematoma: (1) mild, (firstdegree) strain; a tear of a few muscle fibers; minor swelling anddiscomfort with no or only minimal loss of strength and restriction ofmovement; (2) moderate, (second degree) strain; a greater damage ofmuscle fibers with a clear loss of strength, and; (3) severe (thirddegree) strain; a tear extending across the whole muscle belly,resulting in a total loss of muscle function.

[0006] Tearing of the intramuscular blood vessels during muscle straincan often result in a large hematoma. Two different types of hematomasoccur in the injured muscle: intramuscular and intermuscular hematomas.The first type, intramuscular hematomas, is limited in size by theintact muscle fascia. There, the extravasation of blood increases theintramuscular pressure, compressing and limiting the size of thehematoma. Such type of hematoma causes pain and loss of function of themuscle. The second type, intermuscular hematomas, develops when themuscle fascia is ruptured and extravasated blood spreads into theintermuscular spaces without significantly increasing the pressurewithin the muscle. This type of hematoma may not cause significant painif the pressure within the muscle does not increase.

[0007] For treatments of strain injuries, it is critical that theinjured muscle be immobilized, especially during the first two to threedays after the injury, since mobilization of the injured musclesimmediately after the injury often causes re-rupturing at the originalinjury site. A re-rupturing may lead to more severe injuries, delayedhealing and scarring of tissues. Jarvinen et al., Curr Opin Rheumatol,vol 12:155-1-61 (2000).

[0008] Re-rupturing of the damaged site may be avoided by immobilizingthe injured muscle, preferably immediately after the injury.Immobilization allows the newly formed granulation tissue to reachsufficient tensile strength to withstand the forces created bycontracting muscle.

[0009] A known method for immobilization of an injured/strained musclerequires use of a physical restraint or cast. For example, a cervicalcollar can be used to immobilize an injured cervical flexor or extensor.However, the use of a restraint is often cumbersome and uncomfortable.Moreover, for injuries of certain muscle groups, it is not practical orpossible to use a physical restraint. For example, it is very difficultto immobilize a strained upper trapezius or gluteus maximus muscle witha restraint.

[0010] Botulinum Toxin

[0011] The anaerobic, gram positive bacterium Clostridium botulinumproduces a potent polypeptide neurotoxin, botulinum toxin, which causesa neuroparalytic illness in humans and animals referred to as botulism.The spores of Clostridium botulinum are found in soil and can grow inimproperly sterilized and sealed food containers of home basedcanneries, which are the cause of many of the cases of botulism. Theeffects of botulism typically appear 18 to 36 hours after eating thefoodstuffs infected with a Clostridium botulinum culture or spores. Thebotulinum toxin can apparently pass unattenuated through the lining ofthe gut and attack peripheral motor neurons. Symptoms of botulinum toxinintoxication can progress from difficulty walking, swallowing, andspeaking to paralysis of the respiratory muscles and death.

[0012] Botulinum toxin type A (“BoNT/A”) is the most lethal naturalbiological neurotoxin known to man. About 50 picograms of botulinumtoxin (purified neurotoxin complex) serotype A is a LD₅₀ in mice. Oneunit (U) of botulinum toxin is defined as the LD₅₀ upon intraperitonealinjection into female Swiss Webster mice weighing 18-20 grams each.Seven immunologically distinct botulinum neurotoxins have beencharacterized, these being respectively botulinum neurotoxin serotypesA, B, C₁, D, E, F and G each of which is distinguished by neutralizationwith serotype-specific antibodies. The different serotypes of botulinumtoxin vary in the animal species that they affect and in the severityand duration of the paralysis they evoke. For example, it has beendetermined that BoNt/A is 500 times more potent, as measured by the rateof paralysis produced in the rat, than is botulinum toxin serotype B(BoNT/B). Additionally, BoNt/B has been determined to be non-toxic inprimates at a dose of 480 U/kg which is about 12 times the primate LD₅₀for BoNt/A. Botulinum toxin apparently binds with high affinity tocholinergic motor neurons, is translocated into the neuron and blocksthe release of acetylcholine.

[0013] Botulinum toxins have been used in clinical settings for thetreatment of neuromuscular disorders characterized by hyperactiveskeletal muscles. BoNt/A has been approved by the U.S. Food and DrugAdministration for the treatment of blepharospasm, strabismus andhemifacial spasm. Non-serotype A botulinum toxin serotypes apparentlyhave a lower potency and/or a shorter duration of activity as comparedto BoNt/A. Clinical effects of intramuscular of a botulinum toxin, suchas BoNt/A, can be noted in a matter of hours. Thus, it is important tonote that most if not all of the botulinum toxins can, uponintramuscular injection, produce significant muscle paralysis within oneday of the injection, as measured, for example, by the mouse DigitAbduction Score (DAS). Aoki K. R., Preclinical Update on BOTOX(Botulinum Toxin Type A)-Purified Neurotoxin Complex Relative to OtherBotulinum Toxin Preparations, Eur J. Neur 1999, 6 (suppl 4):S3-S10. Thetypical duration of symptomatic relief from a single intramuscularinjection of BoNt/A averages about three months. Botulinum toxins,including botulinum toxin type A, with reduced periods of in vivobiological activity are set forth in co-pending U.S. patent applicationSer. No. 09/620840, which application is incorporated herein byreference in its entirety.

[0014] Although all the botulinum toxins serotypes apparently inhibitrelease of the neurotransmitter acetylcholine at the neuromuscularjunction, they do so by affecting different neurosecretory proteinsand/or cleaving these proteins at different sites. For example,botulinum serotypes A and E both cleave the 25 kiloDalton (kD)synaptosomal associated protein (SNAP-25), but they target differentamino acid sequences within this protein. BoNT/B, D, F and G act onvesicle-associated protein (VAMP, also called synaptobreyin), with eachserotype cleaving the protein at a different site. Finally, botulinumtoxin serotype C₁ (BoNT/C₁) has been shown to cleave both syntaxin andSNAP-25. These differences in mechanism of action may affect therelative potency and/or duration of action of the various botulinumtoxin serotypes.

[0015] Regardless of serotype, the molecular mechanism of toxinintoxication appears to be similar and to involve at least three stepsor stages. In the first step of the process, the toxin binds with highaffinity to the presynaptic membrane of the target neuron through aspecific interaction between the H chain and a cell surface receptor;the receptor is thought to be different for each serotype of botulinumtoxin and for tetanus toxin. The carboxyl end segment of the H chain,H_(c), appears to be important for targeting of the toxin to the cellsurface.

[0016] In the second step, the toxin crosses the plasma membrane of thepoisoned cell. The toxin is first engulfed by the cell throughreceptor-mediated endocytosis, and an endosome containing the toxin isformed. The toxin then escapes the endosome into the cytoplasm of thecell. This last step is thought to be mediated by the amino end segmentof the H chain, H_(N), which triggers a conformational change of thetoxin in response to a pH of about 5.5 or lower. Endosomes are known topossess a proton pump which decreases intra endosomal pH. Theconformational shift exposes hydrophobic residues in the toxin, whichpermits the toxin to embed itself in the endosomal membrane. The toxinthen translocates through the endosomal membrane into the cytosol.

[0017] The last step of the mechanism of botulinum toxin activityappears to involve cleavage of the critical intracellular exocytosisproteins by the L chain. The entire toxic activity of botulinum andtetanus toxins is contained in the L chain of the holotoxin; the L chainis a zinc (Zn++) endopeptidase which selectively cleaves proteinsessential for recognition and docking of neurotransmitter-containingvesicles with the cytoplasmic surface of the plasma membrane, and fusionof the vesicles with the plasma membrane. Tetanus neurotoxin, botulinumtoxin/B/D,/F, and/G cause degradation of synaptobrevin (also calledvesicle-associated membrane protein (VAMP)), a synaptosomal membraneprotein. Most of the VAMP present at the cytosolic surface of thesynaptic vesicle is removed as a result of any one of these cleavageevents. Each toxin specifically cleaves a different bond.

[0018] The molecular weight of the botulinum toxin protein molecule, forall seven of the known botulinum toxin serotypes, is about 150 kD.Interestingly, the botulinum toxins are released by Clostridialbacterium as complexes comprising the 150 kD botulinum toxin proteinmolecule along with associated non-toxin proteins. Thus, the BoNt/Acomplex can be produced by Clostridial bacterium as 900 kD, 500 kD and300 kD forms. BoNT/B and C₁ are apparently produced as only a 500 kDcomplex. BoNT/D is produced as both 300 kD and 500 kD complexes.Finally, BoNT/E and F are produced as only approximately 300 kDcomplexes. The complexes (i.e. molecular weight greater than about 150kD) are believed to contain a non-toxin hemaglutinin protein and anon-toxin and non-toxic nonhemaglutinin protein. These two non-toxinproteins (which along with the botulinum toxin molecule comprise therelevant neurotoxin complex) may act to provide stability againstdenaturation to the botulinum toxin molecule and protection againstdigestive acids when toxin is ingested. Additionally, it is possiblethat the larger (greater than about 150 kD molecular weight) botulinumtoxin complexes may result in a slower rate of diffusion of thebotulinum toxin away from a site of intramuscular injection of abotulinum toxin complex.

[0019] In vitro studies have indicated that botulinum toxin inhibitspotassium cation induced release of both acetylcholine andnorepinephrine from primary cell cultures of brainstem tissue.Additionally, it has been reported that botulinum toxin inhibits theevoked release of both glycine and glutamate in primary cultures ofspinal cord neurons and that in brain synaptosome preparations botulinumtoxin inhibits the release of each of the neurotransmittersacetylcholine, dopamine, norepinephrine, CGRP and glutamate.

[0020] BoNt/A can be obtained by establishing and growing cultures ofClostridium botulinum in a fermenter and then harvesting and purifyingthe fermented mixture in accordance with known procedures. All thebotulinum toxin serotypes are initially synthesized as inactive singlechain proteins which must be cleaved or nicked by proteases to becomeneuroactive. The bacterial strains that make botulinum toxin serotypes Aand G possess endogenous proteases and serotypes A and G can thereforebe recovered from bacterial cultures in predominantly their active form.In contrast, botulinum toxin serotypes C₁, D and E are synthesized bynonproteolytic strains and are therefore typically unactivated whenrecovered from culture. Serotypes B and F are produced by bothproteolytic and nonproteolytic strains and therefore can be recovered ineither the active or inactive form. However, even the proteolyticstrains that produce, for example, the BoNt/B serotype only cleave aportion of the toxin produced. The exact proportion of nicked tounnicked molecules depends on the length of incubation and thetemperature of the culture. Therefore, a certain percentage of anypreparation of, for example, the BoNt/B toxin is likely to be inactive,possibly accounting for the known significantly lower potency of BoNt/Bas compared to BoNt/A. The presence of inactive botulinum toxinmolecules in a clinical preparation will contribute to the overallprotein load of the preparation, which has been linked to increasedantigenicity, without contributing to its clinical efficacy.Additionally, it is known that BoNt/B has, upon intramuscular injection,a shorter duration of activity and is also less potent than BoNt/A atthe same dose level.

[0021] It has been reported that BoNt/A has been used in clinicalsettings as follows:

[0022] (1) about 75-125 units of BOTOX®¹ per intramuscular injection(multiple muscles) to treat cervical dystonia;

[0023] (2) 5-10 units of BOTOX® per intramuscular injection to treatglabellar lines (brow furrows) (5 units injected intramuscularly intothe procerus muscle and 10 units injected intramuscularly into eachcorrugator supercilii muscle);

[0024] (3) about 30-80 units of BOTOX® to treat constipation byintrasphincter injection of the puborectalis muscle;

[0025] (4) about 1-5 units per muscle of intramuscularly injected BOTOX®to treat blepharospasm by injecting the lateral pre-tarsal orbicularisoculi muscle of the upper lid and the lateral pre-tarsal orbicularisoculi of the lower lid.

[0026] (5) to treat strabismus, extraocular muscles have been injectedintramuscularly with between about 1-5 units of BOTOX®, the amountinjected varying based upon both the size of the muscle to be injectedand the extent of muscle paralysis desired (i.e. amount of dioptercorrection desired).

[0027] (6) to treat upper limb spasticity following stroke byintramuscular injections of BOTOX® into five different upper limb flexormuscles, as follows:

[0028] (a) flexor digitorum profundus: 7.5 U to 30 U

[0029] (b) flexor digitorum sublimus: 7.5 U to 30 U

[0030] (c) flexor carpi ulnaris: 10 U to 40 U

[0031] (d) flexor carpi radialis: 15 U to 60 U

[0032] (e) biceps brachii: 50 U to 200 U. Each of the five indicatedmuscles has been injected at the same treatment session, so that thepatient receives from 90 U to 360 U of upper limb flexor muscle BOTOX®by intramuscular injection at each treatment session.

[0033] The success of BoNt/A to treat a variety of clinical conditionshas led to interest in other botulinum toxin serotypes. A study of twocommercially available BoNT/A preparations (BOTOX® and Dysport®) andpreparations of BoNT/B and F (both obtained from Wako Chemicals, Japan)has been carried out to determine the preclinical local muscle weakeningefficacy, safety and antigenic potential. Botulinum toxin preparationswere injected into the head of the right gastrocnemius muscle (0.5 to200.0 units/kg) and muscle weakness was assessed using the mouse digitabduction scoring assay (DAS). ED₅₀ values were calculated from doseresponse curves. Additional mice were given intramuscular injections todetermine LD₅₀ doses. The therapeutic index was calculated as LD₅₀/ED₅₀.Separate groups of mice received hind limb injections of BOTOX® (5.0 to10.0 units/kg) or BoNt/B (50.0 to 400.0 units/kg), and were tested formuscle weakness and increased water consumption, the later being aputative model for dry mouth. Antigenic potential was assessed bymonthly intramuscular injections in rabbits (2.0 or 8.7 Units/kg forBoNt/B or 3.0 Units/kg for BOTOX®). Peak muscle weakness and durationwere dose related for all serotypes. DAS ED₅₀ values (units/kg) were asfollows: BOTOX®: 6.7, Dysport®: 24.7, BoNt/B: 11.8 to 244.0, BoNT/F:4.3. BOTOX® had a longer duration of action than BoNt/B or BoNt/F.Therapeutic index values were as follows: BOTOX®: 10.5, Dysport®: 6.3,BoNt/B: 4.8. Water consumption was greater in mice injected with BoNt/Bthan with BOTOX®, although BoNt/B was less effective at weakeningmuscles. After four months of injections 2 of 4 (where treated with 1.5ng/kg) and 4 of 4 (where treated with 6.5 ng/kg) rabbits developedantibodies against BoNt/B. In a separate study, 0 of 9 BOTOX® treatedrabbits demonstrated antibodies against BoNt/A. DAS results indicaterelative peak potencies of BoNt/A being equal to BoNt/F, and BoNt/Fbeing greater than BoNt/B. With regard to duration of effect, BoNt/A wasgreater than BoNt/B, and BoNt/B duration of effect was greater thanBoNt/F. As shown by the therapeutic index values, the two commercialpreparations of BoNt/A (BOTOX® and Dysport®) are different. Theincreased water consumption behavior observed following hind limbinjection of BoNt/B indicates that clinically significant amounts ofthis serotype entered the murine systemic circulation. The results alsoindicate that in order to achieve efficacy comparable to BoNt/A, it isnecessary to increase doses of the other serotypes examined. Increaseddosage can comprise safety. Furthermore, in rabbits, serotype B was moreantigenic than was BOTOX®, possibly because of the higher protein loadinjected to achieve an effective dose of BoNt/B.

[0034] The tetanus neurotoxin acts mainly in the central nervous system,while botulinum neurotoxin acts at the neuromuscular junction; both actby inhibiting acetylcholine release from the axon of the affected neuroninto the synapse, resulting in paralysis. The effect of intoxication onthe affected neuron is long-lasting and until recently has been thoughtto be irreversible. The tetanus neurotoxin is known to exist in oneimmunologically distinct serotype.

[0035] Acetylcholine

[0036] Typically only a single type of small molecule neurotransmitteris released by each type of neuron in the mammalian nervous system. Theneurotransmitter acetylcholine is secreted by neurons in many areas ofthe brain, but specifically by the large pyramidal cells of the motorcortex, by several different neurons in the basal ganglia, by the motorneurons that innervate the skeletal muscles, by the preganglionicneurons of the autonomic nervous system (both sympathetic andparasympathetic), by the postganglionic neurons of the parasympatheticnervous system, and by some of the postganglionic neurons of thesympathetic nervous system. Essentially, only the postganglionicsympathetic nerve fibers to the sweat glands, the piloerector musclesand a few blood vessels are cholinergic and most of the postganglionicneurons of the sympathetic nervous system release the neurotransmitternorepinephrine. In most instances acetylcholine has an excitatoryeffect. However, acetylcholine is known to have inhibitory effects atsome of the peripheral parasympathetic nerve endings, such as inhibitionof the heart by the vagal nerve.

[0037] The efferent signals of the autonomic nervous system aretransmitted to the body through either the sympathetic nervous system orthe parasympathetic nervous system. The preganglionic neurons of thesympathetic nervous system extend from preganglionic sympathetic neuroncell bodies located in the intermediolateral horn of the spinal cord.The preganglionic sympathetic nerve fibers, extending from the cellbody, synapse with postganglionic neurons located in either aparavertebral sympathetic ganglion or in a prevertebral ganglion. Since,the preganglionic neurons of both the sympathetic and parasympatheticnervous system are cholinergic, application of acetylcholine to theganglia will excite both sympathetic and parasympathetic postganglionicneurons.

[0038] Acetylcholine activates two types of receptors, muscarinic andnicotinic receptors. The muscarinic receptors are found in all effectorcells stimulated by the postganglionic neurons of the parasympatheticnervous system, as well as in those stimulated by the postganglioniccholinergic neurons of the sympathetic nervous system. The nicotinicreceptors are found in the synapses between the preganglionic andpostganglionic neurons of both the sympathetic and parasympathetic. Thenicotinic receptors are also present in many membranes of skeletalmuscle fibers at the neuromuscular junction.

[0039] Acetylcholine is released from cholinergic neurons when small,clear, intracellular vesicles fuse with the presynaptic neuronal cellmembrane. A wide variety of non-neuronal secretory cells, such as,adrenal medulla (as well as the PC12 cell line) and pancreatic isletcells release catecholamines and insulin, respectively, from largedense-core vesicles. The PC12 cell line is a clone of ratpheochromocytoma cells extensively used as a tissue culture model forstudies of sympathoadrenal development. Botulinum toxin inhibits therelease of both types of compounds from both types of cells in vitro,permeabilized (as by electroporation) or by direct injection of thetoxin into the denervated cell. Botulinum toxin is also known to blockrelease of the neurotransmitter glutamate from cortical synaptosomescell cultures.

[0040] A neuromuscular junction is formed in skeletal muscle by theproximity of axons to muscle cells. A signal transmitted through thenervous system results in an action potential at the terminal axon, withactivation of ion channels and resulting release of the neurotransmitteracetylcholine from intraneuronal synaptic vesicles, for example at themotor endplate of the neuromuscular junction. The acetylcholine crossesthe extracellular space to bind with acetylcholine receptor proteins onthe surface of the muscle end plate. Once sufficient binding hasoccurred, an action potential of the muscle cell causes specificmembrane ion channel changes, resulting in muscle cell contraction. Theacetylcholine is then released from the muscle cells and metabolized bycholinesterases in the extracellular space. The metabolites are recycledback into the terminal axon for reprocessing into further acetylcholine.

[0041] As discussed above, the present methods of treating injuredmuscles are still inadequate. There is a need to have improved methodsof treating injured muscles.

SUMMARY

[0042] In accordance with the present invention, an effective method fortreating an injured muscle includes the step of in vivo, localadministration of a therapeutically effective amount of a neurotoxininto or to the vicinity of the injured muscle. The neurotoxin functionsto provide a temporary chemodenervation of the injured muscle and toreduce the muscle's contractions. An objective of the present inventionis therapy is to facility healing and a speedy return to function of aninjured muscle. The injured muscle may be, for example, a strainedmuscle. In one embodiment, the neurotoxin is administeredintramuscularly or subcutaneously. In another embodiment, the step ofadministering a neurotoxin is preceded by and/or followed by physicaltherapy and/or surgery.

[0043] Further in accordance with the invention, the step ofadministering the neurotoxin is immediately after the muscle is injured,or is as soon thereafter as is practical. In one embodiment, theneurotoxin is effective to immobilize or to substantially immobilize theinjured muscle during at least phase 1 and/or phase 2 of the repairprocess of the injured muscle.

[0044] In accordance with the invention, the neurotoxin can include atargeting component, a therapeutic component and a translocationcomponent. The targeting component can bind to a presynaptic motorneuron. In one embodiment, the targeting component can comprise acarboxyl end fragment of a heavy chain of a butyricum toxin, a tetanitoxin, or of a botulinum toxin type A, B, C₁, D, E, F. G or a variantthereof. The therapeutic component can interfere with or modulate therelease of a neurotransmitter from a neuron or its processes. In oneembodiment, the therapeutic component comprises a light chain of abutyricum toxin, a tetani toxin, or of a botulinum toxin type A, B, C₁,D, E, F, G or a variant thereof. The translocation component canfacilitate the transfer of at least a part of the neurotoxin, forexample the therapeutic component, into the cytoplasm of the targetcell. In one embodiment, the translocation component can comprise anamino end fragment of a heavy chain of a butyricum toxin, a tetanitoxin, or of a botulinum toxin type A, B, C₁, D, E, F, G or variantsthereof.

[0045] Still further in accordance with the invention, the neurotoxin isa botulinum toxin type A, B, E and/or F. In a preferred embodiment, theneurotoxin used to treat an injured muscle is botulinum toxin type A. Infact, the use of botulinum toxin type A is preferred because of itscommercial availability, known clinical uses, and successful applicationto treat muscle injury according to the present invention, as disclosedherein. Use of from about 0.1 U/kg to about 30 U/kg of a botulinum toxintype A and from about 1 U/kg to about 150 U/kg of a botulinum toxin typeB is within the scope of a method practiced according to the presentdisclosed invention. With regard to the other botulinum toxin serotypes(including toxin types E and F) the U/kg dosage to be used is within therange of about 0.1 U/kg to about 150 U/kg, as set forth herein.

[0046] Still further in accordance with the invention, the neurotoxincan be recombinantly produced.

[0047] A detailed embodiment of the present invention is a method fortreating (as by promoting the healing of) an injured muscle by in vivo,local administration of a therapeutically effective amount of abotulinum toxin to an injured muscle, thereby treating the injuredmuscle. The botulinum toxin can be botulinum toxin type A.Significantly, the present invention also encompasses a method fortreating pain associated with an injured muscle by in vivo, localadministration of a therapeutically effective amount of a botulinumtoxin to an injured muscle, thereby reducing the pain associated with aninjured muscle.

[0048] Each and every feature described herein, and each and everycombination of two or more of such features, is included within thescope of the present invention provided that the features included insuch a combination are not mutually inconsistent.

[0049] Definitions

[0050] The following definitions are provided and apply herein.

[0051] “About” means approximately or nearly and in the context of anumerical value or range set forth herein means ±10% of the numericalvalue or range recited or claimed.

[0052] “Heavy chain” means the heavy chain of a clostridial neurotoxin.It preferably has a molecular weight of about 100 kDa and may bereferred to herein as H chain or as H.

[0053] “H_(N)” means a fragment (preferably having a molecular weight ofabout 50 kDa) derived from the H chain of a Clostridial neurotoxin whichis approximately equivalent to the amino terminal segment of the Hchain, or the portion corresponding to that fragment in the intact inthe H chain. It is believed to contain the portion of the natural orwild type clostridial neurotoxin involved in the translocation of the Lchain across an intracellular endosomal membrane.

[0054] “H_(C)” means a fragment (about 50 kDa) derived from the H chainof a clostridial neurotoxin which is approximately equivalent to thecarboxyl terminal segment of the H chain, or the portion correspondingto that fragment in the intact H chain. It is believed to be immunogenicand to contain the portion of the natural or wild type Clostridialneurotoxin involved in high affinity, presynaptic binding to motorneurons.

[0055] “Injured muscle” includes a strained, torn or pulled muscle, aswell as a muscle with a contusion (bruise), laceration, ischemia orrupture.

[0056] “Light chain” means the light chain of a clostridial neurotoxin.It preferably has a molecular weight of about 50 kDa, and can bereferred to as L chain, L or as the proteolytic domain (amino acidsequence) of a clostridial neurotoxin. The light chain is believed to beeffective as an inhibitor of neurotransmitter release when it isreleased into a cytoplasm of a target cell.

[0057] “Local administration” means direct administration of apharmaceutical at or to the vicinity of a site on or within an animalbody, at which site a biological effect of the pharmaceutical isdesired. Local administration excludes systemic routes ofadministration, such as intravenous or oral administration.

[0058] “Neurotoxin” means a chemical entity that is capable ofinterfering with or modulating at least one function of a neuron. The“neurotoxin” can be naturally occurring or man-made. Furthermore, the“neurotoxin” can be a small molecule, a large molecule, a polypeptide, aconjugated-polypeptide or mixtures thereof.

[0059] “Variant” means a chemical entity which is slightly differentfrom a parent chemical entity but which still has a biological effect.The biological effect of the variant may be substantially the same orbetter than that of the parent. For example, a variant light chain of abotulinum toxin having at least one amino acid replaced, modified,deleted or added, may have the same or better ability to prevent therelease of neurotransmitter vesicles. Additionally, the biologicaleffect of a variant may be decreased. For example, a variant light chainof a botulinum toxin type A having a leucine-based motif removed mayhave a shorter biological persistence than that of the parent (ornative) botulinum toxin type A light chain.

DESCRIPTION

[0060] In a broad embodiment, an effective method for treating aninjured muscle according to the present invention can include the stepof locally administering a therapeutically effective amount of aneurotoxin into an injured muscle. Preferably, the injured muscle is astrained muscle.

[0061] A strain injury of the skeletal muscle may be classified as ashearing injury. In shearing injury, not only the myofibers but also themysial sheaths are torn. Almost immediately after the injury of themuscle, a repair process of muscle begins. The repair process of theshearing injury may be divided into three phases.

[0062] Phase 1 is the destruction phase, which is characterized byhematoma formation, myofiber necrosis, and inflammatory cell reaction.The site of rupture of an otherwise healthy muscle often occurs close toits distal myotendinous junction (MTJ) after a strain. The rupturedmyofibers contract and a gap is formed between the stumps. Becauseskeletal muscle is richly vascularized, hemorrhage from the torn vesselsis inescapable and the gap becomes filled with a hematoma, laterreplaced by scar tissue. In shearing injuries the mechanical force tearsthe entire myofiber, damaging the myofiber plasma membrane and leavingsarcoplasm open at the ends of the stumps. Because myofibers are verylong, string-like cells, the necrosis initiated at this site extends allalong the whole length of the ruptured myofiber. The blood vessels arealso torn in shearing injuries; thus, blood-borne inflammatory cellsgain immediate access to the injury site to induce an inflammation.Phase 1 persists for about 2 to 3 days following the injury.

[0063] Phase 2 is the repair phase, which consists of phagocycosis ofthe necrotized tissue, regeneration of the myofibers, production ofconnective tissue scar, and capillary ingrowth. The key step in theregeneration of injured muscle tissue is the vascularization of theinjured area. The restoration of vascular supply is a necessary for theregeneration of an injured muscle. The new capillaries sprout fromsurviving trunks of blood vessels and pierce coward the center ofinjured area. These new capillaries help provide adequate oxygen supplyto the regenerating area.

[0064] Phase 3 is the remodeling phase, which consists of maturation ofthe regenerated myofibers, contraction and reorganization of the scartissue, and restoration of the functional capacity of the repairedmuscle. Phase 2 (repair) and 3 (remodeling) often occur simultaneouslyand persists for about 2 days to about six weeks following phase 1.

[0065] In one embodiment of the present invention, the neurotoxin islocally administered, preferably intramuscularly, to immobilize theinjured muscle to facilitate healing. Local administration of aneurotoxin according to the present disclosed invention can also reducethe pain experienced due to a muscle injury. Preferably, theadministration of the neurotoxin is immediately at the time of injury orclosely thereafter. In one preferred embodiment, the neurotoxin iseffective to immobilize the injured muscle during the destruction phase(phase 1) to prevent re-rupturing of the muscle.

[0066] Without wishing to limit the invention to any particular theoryof mechanism of operation; it is believed that mobilization during therepair and/or remodeling phases is beneficial in that such mobilizationinduces more rapid and intensive capillary ingrowth to the injured area,as well as better muscle fiber regeneration and orientation. Therefore,in one embodiment, the immobilizing effect of the neurotoxin is absentduring the repair phase (phase 2) and/or remodeling phase (phase 3). Ina more preferred embodiment, the neurotoxin is administered and iseffective to immobilize the injured muscle during phase 1, but notduring phases 2 and 3 of the repair process. For example, if theneurotoxin is injected, preferably intramuscularly, immediately to themuscle following an injury, it is preferable that the neurotoxinimmobilizes the injured muscle for about 3 days after the time ofadministration. Alternatively, the neurotoxin can have itsimmobilization effect only up to the point where the patient experienceslittle or no pain in the use of the injured muscle in basic movements.When this critical point is reached, the patient should be encouraged tostart active, progressive mobilization.

[0067] In another embodiment of the present invention, the neurotoxin iseffective to immobilize the injured muscle for all of the phase 1-3periods and for a subsequent muscle injury recovery period thereafter.

[0068] Neurotoxins, such as certain of the botulinum toxins, which canrequire from less than about one day to about seven days to exhibitsignificant clinical muscle paralysis effect and/or and where the muscleparalysis effect is sustained post injection for a period of severalmonths, are within the scope of the present invention, as suchneurotoxins can be used to treat relatively serious or long lastingmuscle injuries or where a long period of muscle immobilization isindicated for proper healing.

[0069] In a broad embodiment, the neurotoxin is a neuromuscular blockingagent. Table 1 shows a non-limiting list of neuromuscular blockingagents and their potential site of actions. In an embodiment,neuromuscular blocking agents having the ability to immobilize muscles,preferably injured muscles, for at least about 5 days, and preferablyfor at least about 3 days are administered to treat injured muscles. Ina preferred embodiment of the present invention, the neurotoxin is abotulinum toxin because of the known uses and clinical safety of abotulinum toxin, such as botulinum toxin type E to treat muscledisorders, such as muscle spasms. In a particularly preferred embodimentof the present invention, especially for severe, or third degree muscleinjuries, the locally administered botulinum toxin is a botulinum toxintype E. Botulinum toxin type A can also be used in both theseembodiments. TABLE 1 Site of Action Relative to Compound NMJPharmacological Class Acetylcholine Synaptic ACh Esterase InducersEsterase Inducers Aconitine Presynaptic Sodium Channel ActivatorAdenoregulin Presynaptic Adenosine Receptor Regulator (from the frogPhyllomedeusa bicolor) Adenosine Agonist Pre & Post Adenosine SynapticAdenosine Pre & Post Adenosine Antagonist Synaptic Adenosine Pre & PostAdenosine Synaptic Regulating Agent Adrenergics Presynaptic AlphaAdrenergic Anatoxin-A Postsynaptic AChR Agonist Antiepileptics CNSAntiepileptics Antisense Pre & Post Antisense technology for Synapticproteins or messages important in specific neurotransmitter release,receptor production. Anxiolytics CNS Anxiolytics Antiepileptic AtacuriumPostsynaptic AChR Antagonist Nondepolarizing muscle relaxant Atracuriumbesylate Postsynaptic AChR Antagonist (Tracurium) Nondepolarizing musclerelaxant Baclofen Presynaptic GABA analog (Lioresal .RTM., Geigy;Intrathecal Medtronic Neurological generic, Athena Biocraft, WarnerChilcott Bacterial, Plant and Fungal Products Batrachotoxin PresynapticSodium Channel Activator Benzylpiperidines Synaptic Cleft ACh EsteraseInhibitors (nontraditional) Botanical Pre and Post varies NeurotoxinsSynaptic as well as Synaptic Cleft Bungarotoxin-β Presynaptic PLA2 andvoltage sensitive (β-BuTX) potassium channel blocker. Snake toxin fromBungarus multicinctus. Bupivacain Pre and Post Local Anesthetic SynapticMyotoxin Captopril Presynaptic Antihypertensive (Capoten .RTM., ACEInhibitor Squibb; Capzide zinc endopeptidase inhibitor .RTM., Squibb)Choline + acetyl Pre Synaptic CAT Inhibitors transferase inhibitorsCholinesterase Synaptic Cleft ACh Esterase Inhibitors InhibitorsCiguatoxins Presynaptic Sodium Channel Conotoxin MI Postsynaptic (alphaConotoxin) ACbR Antagonist Conotoxin-.mu. GIIIA Na+channel blocker(mu-CT) Conotoxin-.OMEGA. GVIA Ca2+ channel blocker in (omega-CT)neutrons only Curare Postsynaptic AChR Antagonist NondepolarizingDantrolene Sodium Postsynaptic Skeletal Muscle Relaxant (Dantrium, P &G) Dauricine Post Synaptic AChR antagonist Decamethonium PresynapticGanglionic blocker Bromide Dendrotoxin Pre and Post Potassium Channelblocker Synaptic Diaminopyridine Presynaptic Botulinum toxinintoxication (3-DAP) Reversal Diazepam CNS Anxiolytic DoxacuriumPostaynaptic AChR Antagonist chloride Nondepolarizing muscle relaxant(Nuromax .RTM., Burroughs Wellcome) Doxorubicin Postsynaptic Myotoxin(Adriamyocin, Chemo Myectomy Adria; Rubex, Immunex; Cetus Onoclogy)Epibatidine Postsynaptic AChR Agonist Dihydtochloride FelbamatePresynaptic Antiepileptic (Felbatol, Carter- CNS Wallace lic toSchering-Plough) Foroxymithine Presynaptic Angiotensin I ConvertingEnzyme inhibitor Gabapentin Presynaptic Antiepileptic (Neurontin, Parke-CNS GABA Ahalog Davis) Gallamine Postsynaptic AChR AntagonistGrayantoxin Presynaptic Sodium Channel Activator HexahydroazepinylPresynaptic ACh Releaser Acetamides and other chemical classes HuperzineA Synaptic Cleft ACh Esterase Inhibitor Insect Venoms Ion Channel Preand Post Channel Blockers Blockers Synaptic Ion Channel Pre and PostChannel Stimulants Stimulants Synaptic Latrotoxin-α Presynaptic CalciumIonophore black widow spider venom component Lidocaine, PresynapticLocal Anesthetics procaine, mepivacain, etc. Linopirdine Presynaptic AChRelease Enhancer (DuP 996, Dupont Merck) Lophotoxin and PostsynapticAChR Antagonist analogs Irreversible Marine Natural ProductsMethocarbamol CNS Depression, (Robaxin, Robins muscle relaxation. Co.)Methyl lycaconitine Mivacurium Postsynaptic AChR Antagonist chlorideNondepolarizing muscle relaxant (Mivacro .RTM., BW- BW109OU, BurroughsWellcome) Modified Pre Synaptic ACh Release Inhibitor Clostridial ToxinsMonoclonal receptor, agrin, antibodies against neurotransmitters, plasmaNMJ components membrane components, inactivating enzymes, etc.Muscarinic Agonist Pre and Post Muscarinic and Antagonists Synaptic, CNSAgonist Antagonist Neosaxitoxin Presynaptic Sodium Channel BlockerNeosurugatoxin Autonomic Ganglionic AChR Blocker. (no effect @ NMJ)Neuromuscular Postsynaptic AChR Antagonists Blocking Agents AChRDepolarizing Neurotoxins from Pre and Post varies reptile, insects,Synaptic as and other sources well as Synaptic Cleft PancuroniumPostsynaptic AChR Antagonist Bromide Nondepolarizing muscle relaxant(Organon) Pancuronium-3-OH Postsynaptic AChR Antagonist metabolitesNondepolarizing muscle relaxant (Organon) Papverine HCl Smooth MuscleRelaxants (30 mg/ml) Physostigmine and Synaptic Cleft ACh Esteraseinhibitor Analogs Pipercuronium Postsynaptic AChR Antagonist (Arduan,Organon) Nondepolarizing muscle relaxant Presynaptic Nerve Pre Synapticany extra or intraneuronal Terminal Recpetors recpetors on nerveterminal Short Neurotoxin Postsynaptic AChR Antagonist alphaβ-Bungarotoxin Presynaptic Snake toxin from Bungarus (β-BuTX)multicinctus. Succinylcholine Postsynaptic AChR Receptor Agonistchloride Depolarizing skeletal muscle (Anectine, relaxant BurroughsWellcome) Tetanus Toxin Presynaptic EAA release inhibitor Tetanus ToxinPresynaptic Transporter Tetrahydroamino- Synaptic Cleft ACh EsteraseInhibitor acridine (THA) Tetrodoxtoxin Pre and Post Sodium ChannelBlocker Synaptic Tiagabine CNS Antiepileptic (Novo Nordisk) GABA uptakeinhibitor Transglutaminase Pre and Post Enzyme inhibitors or Synapticinduction prevention Valium diazepaxn CNS Anxiolytic VecuroniumPostsynaptic AChR Antagonist (Norcuron, Nondepolarizing muscle relaxantOrganon) Vecuronium-3-OH Postsynaptic AChR Antagonist metabolitesNondepolarizing muscle relaxant (Organon) Veratridine Presynaptic SodiumChannel Activator Vigabatrin Presynaptic Antiepileptic (Sabril, MarionCNS GABA metabolism inhibitor Merrell Dow) (irreversible) VesamicolPresynaptic ACh Vesicle transport inhibitor and other drugs with thesame mechanism. Zinc Endopeptidase Pre Synaptic Enyzmes. and otherproteases reduce neurotransmitter release delivered by Botulinum toxinor tetanus toxin transporter

[0070] In a broad embodiment, the neurotoxin can comprise a targetingcomponent, a therapeutic component and a translocation component. Thetargeting component can bind to a presynaptic motor neuron. In oneembodiment, the targeting component can comprise a carboxyl end fragmentof a heavy chain of a butyricum toxin, a tetani toxin, a botulinum toxintype A, B, C1, D, E, F, G or a variant thereof. In a preferredembodiment, the targeting component can include a carboxyl end fragmentof a botulinum toxin type A.

[0071] The therapeutic component can substantially interfere with ormodulate the release of neurotransmitters from a cell or its processes.In one embodiment, the therapeutic component comprises a light chain ofa butyricum toxin, a tetani toxin, a botulinum toxin type A, B, C₁, D,E, F, G or a variant thereof. In a preferred embodiment, the therapeuticcomponent may include a light chain of a botulinum toxin type which hasa short biological persistence, for example less than about 5 days,preferably less than about 3 days. Preferably, such light chain can be alight chain of a botulinum toxin type E or F. Alternately, the lightchain can be a light chain of a botulinum toxin type A.

[0072] The translocation component can facilitate the transfer of atleast a part of the neurotoxin, for example the therapeutic componentinto the cytoplasm of the target cell. In one embodiment, thetranslocation component comprises an amino end fragment of a heavy chainof a butyricum toxin, a tetani toxin, a botulinum toxin type A, B, C₁,D, E, F, G or variants thereof. In a preferred embodiment, thetranslocation component comprises an amino end fragment of a heavy chainof a botulinum toxin type A.

[0073] In one embodiment, the targeting component comprises a carboxylend fragment of a heavy chain of a botulinum toxin type E or F, thetherapeutic component comprises a light chain of a botulinum toxin typeE or F and the translocation component comprises an amine end fragmentof a heavy chain of a botulinum toxin type E or F. In a preferredembodiment, the neurotoxin comprises a botulinum toxin type E. Inanother preferred embodiment, the neurotoxin comprises a botulinum toxintype F. In yet another embodiment, the neurotoxin comprises a mixture ofbotulinum toxin type E and F.

[0074] In one embodiment, the targeting component comprises a carboxylend fragment of a heavy chain of a botulinum toxin type A, thetherapeutic component comprises a light chain of a botulinum toxin typeA and the translocation component comprises an amine end fragment of aheavy chain of a botulinum toxin type A. In a preferred embodiment, theneurotoxin of the present invention comprises a botulinum toxin type A.A suitable botulinum toxin type A to use herein is BOTOX® (Allergan,Inc., Irvine, Calif.)

[0075] Although the neurotoxins of the present invention treats injuredmuscles by immobilizing them, in one embodiment, the neurotoxin may alsobe administered to injured muscles to reduce pain and/or spasm. Inanother embodiment, the neurotoxin is able to immobilize the injuredmuscle and to reduce pain associated with that injured muscle. In apreferred embodiment, a neurotoxin, for example a botulinum toxin typeE, pr most preferably type A, is administered to a strained muscle toimmobilize the muscle and/or to reduce pain associated with that muscle.

[0076] Of course, an ordinarily skilled medical provider can determinethe appropriate dose and frequency of administration(s) to achieve anoptimum clinical result. That is, one of ordinary skill in medicinewould be able to administer the appropriate amount of the neuromuscularblocking agent at the appropriate time(s) to effectively immobilize theinjured muscle(s). The dose of the neurotoxin to be administered dependsupon a variety of factors, including the size of the muscle, theseverity of the muscle injury. In a preferred embodiment, the dose ofthe neurotoxin administered immobilizes the injured muscle(s) for nolonger than the duration of phase 1 of the repair process. In thevarious methods of the present invention, from about 0.1 U/kg to about15 U/kg, of botulinum toxin type A can be administered to the injuredmuscle. Preferably, about 1 U/kg to about 20 U/kg of botulinum toxintype A may be administered to the injured muscle. Use of from about 0.1U/kg to about 30 U/kg of a botulinum toxin type A and from about 1 U/kgto about 150 U/kg of a botulinum toxin type B is within the scope of amethod practiced according to the present disclosed invention. Withregard to the other botulinum toxin serotypes (including toxin types Eand F) the U/kg dosage to be use d is within the range of about 0.1 U/kgto about 150 U/kg, as set forth herein.

[0077] Although intramuscular injection is the preferred route ofadministration, other routes of local administration are available, suchas subcutaneous administration.

[0078] In another broad embodiment, the method of treating injuredmuscle according to this invention further includes other stepsdescribed below. These other steps may be taken prior to, in conjunctionwith or following the step of administering a neurotoxin, preferably tothe injured muscle. For example, the present recommended treatment forstrained muscle includes resting, icing, compression and elevating.These four steps (or procedures) have the same objective. They minimizebleeding from ruptured blood vessels to rupture site. This will preventthe formation of a large hematoma, which has a direct impact on the sizeof scar tissue at the end of the regeneration. A small hematoma and thelimitation of interstitial edema accumulation on the rupture site alsoshorten the ischemic period in the granulation tissue, which in turnaccelerates regeneration.

[0079] Other additional steps may be employed in the treatment ofinjured muscles. In one embodiment, the additional step include anadministration of nonsteroidal anti-inflammatory drugs (NSAIDs),therapeutic ultrasound, hyperbaric oxygen, and in severe injuries,surgery may also be employed. NSAIDs should be a part of early treatmentand should he started immediately after the injury. Short-term use ofNSAIDs in the early phase of healing decreases the inflammatory cellreaction, and has no adverse effects on tensile or contractileproperties of injured muscle.

[0080] In another embodiment, the additional step includes the use oftherapeutic ultrasound. Therapeutic ultrasound is widely recommended andused in the treatment of muscle strains. It is thought that therapeuticultrasound promotes the proliferation phase of myoregeneration.

[0081] In another embodiment, the additional step includes the use ofhyperbaric oxygen. It is known that hyperbaric oxygen therapy in rabbitsduring the early phase of the repair substantially improves the finaloutcome. It is believed that such hyperbaric oxygen therapy in othermammals, for example human beings, may be helpful, such as by speedingup muscle regeneration.

[0082] In another embodiment, the additional step includes surgicalintervention. Surgical treatment of muscle injuries should be reservedfor the most serious injuries, because in most cases conservativetreatment results in a good outcome. Surgical treatment is indicatedonly in cases of (1) large intramuscular hematomas, (2) third-degreestrains or tears of muscles with few or no agonise muscles, and (3)second-degree strains, if more than half of the muscle belly is torn.

[0083] In another broad aspect of this invention, recombinant techniquesare used to produce at least one of the components of the neurotoxins.The technique includes steps of obtaining genetic materials from eitherDNA cloned from natural sources, or synthetic oligonucleotide sequences,which have codes for one of the components, for example the therapeutic,translocation and/or targeting component(s). The genetic constructs areincorporated into host cells for amplification by first fusing thegenetic constructs with a cloning vectors, such as phages or plasmids.Then the cloning vectors are inserted into hosts, preferably E. coli's.Following the expressions of the recombinant genes in host cells, theresultant proteins can be isolated using conventional techniques. Theprotein expressed may comprise all three components of the neurotoxin.For example, the protein expressed may include a light chain ofbotulinum toxin type E (the therapeutic component), a heavy chain,preferably the H_(N), of a botulinum toxin type B (the translocationcomponent), and an H_(c) of botulinum toxin type A, which selectivelybinds to the motor neurons. In one embodiment, the protein expressed mayinclude less than all three components of the neurotoxin. In such case,the components may be chemically joined using techniques known in theart.

[0084] There can be many advantages to producing these neurotoxinsrecombinantly. For example, production of neurotoxin from anaerobicClostridium cultures is a cumbersome and time-consuming processincluding a multi-step purification protocol involving several proteinprecipitation steps and either prolonged and repeated crystallization ofthe toxin or several stages of column chromatography. Significantly, thehigh toxicity of the product dictates that the procedure must beperformed under strict containment (BL-3). During the fermentationprocess, the folded single-chain neurotoxins are activated by endogenousClostridial proteases through a process termed nicking. This involvesthe removal of approximately 10 amino acid residues from thesingle-chain to create the dichain form in which the two chains remaincovalently linked through the intrachain disulfide bond.

[0085] The nicked neurotoxin is much more active than the unnicked form.The amount and precise location of nicking varies with the serotypes ofthe bacteria producing the toxin. The differences in single-chainneurotoxin activation and, hence, the yield of nicked toxin, are due tovariations in the type and amounts of proteolytic activity produced by agiven strain. For example, greater than 99% of Clostridial botulinumtype A single-chain neurotoxin is activated by the Hall A Clostridialbotulinum strain, whereas type B and E strains produce toxins with loweramounts of activation (0 to 75% depending upon the fermentation time).Thus, the high toxicity of the mature neurotoxin plays a major part inthe commercial manufacture of neurotoxins as therapeutic neurotoxins.

[0086] The degree of activation of engineered Clostridial toxins is,therefore, an important consideration for manufacture of thesematerials. It would be a major advantage if neurotoxins such asbotulinum toxin and tetanus toxin could be expressed, recombinantly, inhigh yield in rapidly-growing bacteria (such as heterologous E. colicells) as relatively non-toxic single-chains (or single chains havingreduced toxic activity) which are safe, easy to isolate and simple toconvert to the fully-active form.

[0087] With safety being a prime concern, previous work has concentratedon the expression in E.coli and purification of individual H and Lchains of tetanus and botulinum toxins; these isolated chains are, bythemselves, non-toxic; see Li et al., Biochemistry 33:7014-7020 (1994);Zhou et al., Biochemistry 34:15175-15181 (1995), hereby incorporated byreference herein. Following the separate production of these peptidechains and under strictly controlled conditions the H and L subunits canbe combined by oxidative disulphide linkage to form the neuroparalyticdi-chains.

[0088] The following non-limiting examples provide preferred methods oftreating injured muscles and producing recombinant neurotoxins,preferably botulinum toxins. The methods of producing recombinantbotulinum toxins described in the below Examples 4-8 are drawn from andare similar to those described in Dolly et al. International PatentApplication No. WO 95/32738, the disclosure of which is incorporated inits entirety herein by reference.

EXAMPLE 1 Treatment of a Ruptured Biceps Tendon

[0089] Ruptures of the biceps brachii commonly occur at the proximal endand involve the long head of the biceps. The muscle may rupture at thedistal insertion onto the radius, but is rare. Most often, rupturesoccur in adults older than age 40 years who have a long history ofshoulder pain secondary to an impingement syndrome. Over time, thetendon becomes frayed and weak, and ultimately ruptures, partially orentirely. Regardless, the rupture is often caused by a trivial event.These ruptures are usually associated with a rotator cuff tear,especially among the elderly.

[0090] A 45 year old man presents with a bulge in the lower arm afterlifting heavy boxes. He reports a history of sudden sharp pain in theupper arm, often accompanied by an audible snap. The man is diagnosed ashaving a ruptured biceps tendon and is at the beginning of phase 1 ofthe repair process. The rupture may be classified as a mild seconddegree strain.

[0091] The patient is treated by a bolus injection of between about 0.1U/kg to about 25 U/kg of a neurotoxin intramuscularly to the biceps.Preferably the neurotoxin is botulinum toxin type E and/or F, morepreferably type A. The particular dose and frequency of administrationsdepend upon a variety of factors, and are to be determined by thetreating physician. The patient is further instructed to rest and applyice and compression to the biceps. Within about three days after theadministration of the neurotoxin, the patient is able to bend his arm.Also, after about three days, the patient experiences a reduction ininflammation, which is a sign that the patient is entering into phase 2and 3 of the repair process. The patient also experiences a significantpain reduction. Local administration of from about 10 units to about 200units of botulinum toxin type A can also be used for long term (2-4months) muscle immobilization and pain reduction.

EXAMPLE 2 Extensor Mechanism Rupture

[0092] Rupture of the extensor mechanism of the knee occurs in one oftwo ways: in younger patients as a result of a sudden or violent force(such as jumping, heavy lifting); and in older patients as a result ofrelatively trivial force. In either group, there may have been someprior arching. This condition affects older patients who have typicallybeen somewhat sedentary and have suddenly increased their activitylevel, or patient who have had some preexisting or co-existing conditionsuch as diabetes mellitus, rheumatoid arthritis, and other systemicinflammatory disorders, or prior knee surgery.

[0093] A 22 year old female soccer player presents with an inability toextend her knee. The patient also is also unable to do straight legraise, but is able to walk if she keeps a hand on her thigh and maintainher knee in extension. A plain radiograph shows that the patella is in alower than usual location. The patient is diagnosed with a severerupture of the quadriceps.

[0094] After determining the injury is severe (third degree), thepatient agrees to undergo reparative surgery. Post-operationally, thepatient is treated by a bolus injection of between about 0.1 U/kg toabout 25 U/kg of a neurotoxin (such as about 10 units to about 400 unitsof botulinum toxin type A) intramuscularly to the quadriceps. Preferablythe neurotoxin is botulinum toxin type A. The particular dose andfrequency of administrations depend upon a variety of factors, and areto be determined by the treating physician. The patient is furtherinstructed to rest and apply ice and compression to the quadriceps.Within about 15 days after the administration of the neurotoxin, gradualmovement and activity of the injured muscle is possible. The patient isthen encouraged to gently move the recovering muscle to strengthen itand the surrounding muscles. As the toxin effect wears off some more,the patient would then have the ability to rapidly participate in aphysical therapy program or resume the general activity and/or sport. Ifthis patient depended upon this sport for her livelihood, botulinumtoxin therapy would facilitate her early return to this activity. Localadministration of from about 10 units to about 200 units of botulinumtoxin type A can be used for long term (2-4 months) muscleimmobilization.

EXAMPLE 3 Treatment of Shin Splints

[0095] Runners commonly experience shin splits in the lower limb whichcauses pain and restricts this activity. The lower leg pain resultingfrom shin splits is caused by very small tears in the leg muscles attheir point of attachment to the shin. There are two types: 1. Anteriorshin splints occur in the front portion of the shinbone (tibia). 2.Posterior shin splints occur on the inside (medial) part of the legalong the tibia.

[0096] Anterior shin splints are due to muscle imbalances, insufficientshock absorption or toe running. Excessive pronation contributes to bothanterior and posterior shin splints.

[0097] In treating strained muscle, such as a shin splint, five stepsare recommended: (1) Protect the injured muscle from further injury byusing splints, pads and/or crutches; (2) Restrict activity, usually for48 to 72 hours to allow the healing process to begin. The administrationof a short acting botulinum toxin type E or F or a botulinum toxin typeA modified so as to reduce the period of in vivo biological activity(i.e. a shorter period of flaccid muscle paralysis) of the type A toxin.Suitable botulinum toxins, including botulinum toxin type A, withreduced periods of in vivo biological activity suitable for use hereinare set forth in co-pending U.S. patent application Ser. No. 09/620840,which application is incorporated herein by reference in its entirety.In more severe strains restriction of activity can last for weeks tomonths. With a longer required restriction of activity, anadministration of a longer acting botulinum toxin, for example(unmodified) botulinum toxin type B, or more preferably, type A toxin,can be appropriate. Without this treatment, patients could experienceweeks of restricted activity. As the healing process begins, gentlemotion and movement of the affected muscle is advised; (3) Ice should beapplied for 15-20 minutes every hour; (4) Compression such as elasticbandage should be kept on between icing; and (5) Elevate the injuredarea to minimize swelling.

EXAMPLE 4 Subcloning the BoNT/A-L Chain Gene

[0098] This Example describes the methods to clone the polynucleotidesequence encoding the BoNT/A-L chain. The DNA sequence encoding theBoNT/A-L chain is amplified by a PCR protocol that employs syntheticoligonucleotides having the sequences, 5′-AAAGGCCTTTTGTTAATAAACAA-3′(SEQ ID#1) and 5′-GGAATTCTTACTTATTGTATCCTTTA-3′(SEQ ID#2). Use of theseprimers allows the introduction of Stu I and EcoR I restriction sitesinto the 5′ and 3′ ends of the BoNT/A-L chain gene fragment,respectively. These restriction sites are subsequently used tofacilitate unidirectional subcloning of the amplification products.Additionally, these primers introduce a stop codon at the C-terminus ofthe L chain coding sequence. Chromosomal DNA from C. botulinum (strain63 A) serves as a template in the amplification reaction.

[0099] The PCR amplification is performed in a 100 μl volume containing10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 0.2 mM of eachdeoxynucleotide triphosphate (dNTP), 50 pmol of each primer, 200 ng ofgenomic DNA and 2.5 units of Taq-polymerase (Promega). The reactionmixture is subjected to 35 cycles of denaturation (1 minute at 94° C.),annealing (2 minutes at 37° C.) and polymerization (2 minutes at 72°C.). Finally, the reaction is extended for an additional 5 minutes at72° C.

[0100] The PCR amplification product is digested with Stu I and EcoR I,purified by agarose gel electrophoresis, and ligated into Sma I and EcoRI digested pBluescript II SK* to yield the plasmid, pSAL. Bacterialtransformants harboring this plasmid are isolated by standardprocedures. The identity of the cloned L chain polynucleotide isconfirmed by double stranded plasmid sequencing using SEQUENASE (UnitedStates Biochemicals) according to the manufacturer's instructions.Synthetic oligonucleotide sequencing primers are prepared as necessaryto achieve overlapping sequencing runs. The cloned sequence is found tobe identical to the sequence disclosed by Binz, et al., in J. Biol.Chem. 265:9153 (1990), and Thompson et al., in Eur. J. Biochem. 189:73(1990).

[0101] Site-directed mutants designed to compromise the enzymaticactivity of the BoNT/A-L chain can also be created.

EXAMPLE 5 Expression of the Botulinum Toxin Type A-L (BoNt/A-L) ChainFusion Proteins

[0102] This Example describes the methods to verify expression of thewild-type L chains, which may serve as a therapeutic component, inbacteria harboring the pCA-L plasmids. Well isolated bacterial coloniesharboring either pCAL are used to inoculate L-broth containing 100 μg/mlampicillin and 2% (w/v) glucose, and grown overnight with shaking at 30°C. The overnight cultures are diluted 1:10 into fresh L-broth containing100 μg/ml of ampicillin and incubated for 2 hours. Fusion proteinexpression is induced by addition of IPTG to a final concentration of0.1 mM. After an additional 4 hour incubation at 30° C., bacteria arecollected by centrifugation at 6,000×g for 10 minutes.

[0103] A small-scale SDS-PAGE analysis confirmed the presence of a 90kDa protein band in samples derived from IPTG-induced bacteria. ThisM_(r) is consistent with the predicted size of a fusion protein havingMBP (˜40 kDa) and BoNT/A-L chain (˜50 kDa) components. Furthermore, whencompared with samples isolated from control cultures, the IPTG-inducedclones contained substantially larger amounts of the fusion protein.

[0104] The presence of the desired fusion proteins in IPTG-inducedbacterial extracts is also confirmed by Western blotting using thepolyclonal anti-L chain probe described by Cenci di Bello et al., inEur. J. Biochem. 219:161 (1993). Reactive bands on PVDF membranes(Pharmacia; Milton Keynes, UK) are visualized using an anti-rabbitimmunoglobulin conjugated to horseradish peroxidase (Bio-Rad; HemelHempstead, UK) and the ECL detection system (Amersham, UK). Westernblotting results confirmed the presence of the dominant fusion proteintogether with several faint bands corresponding to proteins of lowerM_(r) than the fully sized fusion protein. This observation suggestedthat limited degradation of the fusion protein occurred in the bacteriaor during the isolation procedure. Neither the use of 1 mM nor 10 mMbenzamidine (Sigma; Poole, UK) during the isolation procedure eliminatedthis proteolytic breakdown.

[0105] The yield of intact fusion protein isolated by the aboveprocedure remained fully adequate for ell procedures described herein.Based on estimates from stained SDS-PAGE gels, the bacterial clonesinduced with IPTG yielded 5-10 mg of total MBP-wild-type or mutant Lchain fusion protein per liter of culture. Thus, the method of producingBoNT/A-L chain fusion proteins disclosed herein is highly efficient,despite any limited proteolysis that did occur.

[0106] The MBP-L chain fusion proteins encoded by the pCAL andpCAL-TyrU7 expression plasmids are purified from bacteria by amyloseaffinity chromatography. Recombinant wild-type or mutant L chains arethen separated from the sugar binding domains of the fusion proteins bysite-specific cleavage with Factor X₂. This cleavage procedure yieldedfree MBP, free L chains and a small amount of uncleaved fusion protein.While the resulting L chains present in such mixtures have been shown topossess the desired activities, we have also employed an additionalpurification step. Accordingly, the mixture of cleavage products isapplied to a second amylose affinity column that bound both the MBP anduncleaved fusion protein. Free L chains are not retained on the affinitycolumn, and are isolated for use in experiments described below.

EXAMPLE 6 Purification of Fusion Proteins and Isolation of RecombinantBoNT/A-L Chains

[0107] This Example describes a method to produce and purify wild-typerecombinant BoNT/A light chains from bacterial clones. Pellets from 1liter cultures of bacteria expressing the wild-type BoNT/A-L chainproteins are resuspended in column buffer [10 mM Tris-HCl (pH 8.0), 200mM NaCl, 1 mM EGTA and 1 mM DTT] containing 1 mM phenyl-methanesulfonylfluoride (PMSF) and 10 mM benzamidine, and lysed by sonication. Thelysates are cleared by centrifugation at 15,000×g for 15 minutes at 4°C. Supernatants are applied to an amylose affinity column [2×10 cm, 30ml resin] (New England BioLabs; Hitchin, UK). Unbound proteins arewashed from the resin with column buffer until the eluate is free ofprotein as judged by a stable absorbance reading at 280 nm. The boundMBP-L chain fusion protein is subsequently eluted with column buffercontaining 10 mM maltose. Fractions containing the fusion protein arepooled and dialyzed against 20 mM Tris-HCl (pH 8.0) supplemented with150 mM NaCl, 2 mM, CaCl₂ and 1 mM DTT for 72 hours at 4° C.

[0108] Fusion proteins are cleaved with Factor X₂ (Promega; Southampton,UK) at an enzyme:substrate ratio of 1:100 while dialyzing against abuffer of 20 mM Tris-HCl (pH 8.0) supplemented with 150 mM NaCl, 2 mM,CaCl₂ and 1 mM DTT. Dialysis is carried out for 24 hours at 4° C. Themixture of MBP and either wild-type or mutant L chain that resulted fromthe cleavage step is loaded onto a 10 ml amylose column equilibratedwith column buffer. Aliquots of the flow through fractions are preparedfor SDS-PAGE analysis to identify samples containing the L chains.Remaining portions of the flow through fractions are stored at −20° C.Total E. coli extract or the purified proteins are solubilized in SDSsample buffer and subjected to PAGE according to standard procedures.Results of this procedure indicated the recombinant toxin fragmentaccounted for roughly 90% of the protein content of the sample.

[0109] The foregoing results indicates that the approach to creatingMBP-L chain fusion proteins described herein could be used toefficiently produce wild-type and mutant recombinant BoNT/A-L chains.Further, the results demonstrate that recombinant L chains could beseparated from the maltose binding domains of the fusion proteins andpurified thereafter.

[0110] A sensitive antibody-based assay is developed to compare theenzymatic activities of recombinant L chain products and their nativecounterparts. The assay employed an antibody having specificity for theintact C-terminal region of SNAP-25 that corresponded to the BoNT/Acleavage site. Western Blotting of the reaction products of BoNT/Acleavage of SNAP-25 indicated an inability of the antibody to bindSNAP-25 sub-fragments. Thus, the antibody reneurotoxin employed in thefollowing Example detected only intact SNAP-25. The loss of antibodybinding served as an indicator of SNAP-25 proteolysis mediated by addedBoNT/A light chain or recombinant derivatives thereof.

EXAMPLE 7 Evaluation of the Proteolytic Activities of Recombinant LChains Against a SNAP-25 Substrate

[0111] This Example describes a method to demonstrate that both nativeand recombinant BoNT/A-L chains can proteolyze a SNAP-25 substrate. Aquantitative assay is employed to compare the abilities of the wild-typeand their recombinant analogs to cleave a SNAP-25 substrate. Thesubstrate utilized for this assay is obtained by preparing aglutathione-S-transferase (GST)-SNAP-25 fusion protein, containing acleavage site for thrombin, expressed using the pGEX-2T vector andpurified by affinity chromatography on glutathione agarose. The SNAP-25is then cleaved from the fusion protein using thrombin in 50 mM Tris-HCl(pH 7.5) containing 150 mM NaCl and 2.5 mM CaCl₂ (Smith et al., Gene67:31 (1988)) at an enzyme:substrate ratio of 1:100. Uncleaved fusionprotein and the cleaved glutathione-binding domain bound to the gel. Therecombinant SNAP-25 protein is eluted with the latter buffer anddialyzed against 100 mM HEPES (pH 7.5) for 24 hours at 4° C. The totalprotein concentration is determined by routine methods.

[0112] Rabbit polyclonal antibodies specific for the C-terminal regionof SNAP-25 are raised against a synthetic peptide having the amino acidsequence, CANQRATKMLGSG (SEQ ID#3). This peptide corresponded toresidues 195 to 206 of the synaptic plasma membrane protein and anN-terminal cysteine residue not found in native SNAP-25. The syntheticpeptide is conjugated to bovine serum albumin (BSA) (Sigma; Poole, UK)using maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) as across-linking neurotoxin (Sigma; Poole, UK) to improve antigenicity (Liuet al., Biochemistry 18:690 (1979)1. Affinity purification of theanti-peptide antibodies is carried out using a column having theantigenic peptide conjugated via its N-terminal cysteine residue to anaminoalkyl agarose resin (Bio-Rad; Hemel Hempstead, UK), activated withiodoacetic acid using the cross-linker ethyl 3-(3-dimethytpropyl)carbodiumide. After successive washes of the column with a buffercontaining 25 mM Tris-HCl (pH 7.4) and 150 mM NaCl, the peptide-specificantibodies are eluted using a solution of 100 mM glycine (pH 2.5) and200 mM NaCl, and collected in tubes containing 0.2 ml of 1 M Tris-HCl(pH 8.0) neutralizing buffer.

[0113] All recombinant preparations containing wild-type L chain aredialyzed overnight at 4° C. into 100 mM HEPES (pH 7.5) containing 0.02%Lubrol and 10 μM zinc acetate before assessing their enzymaticactivities. BoNT/A, previously reduced with 20 mM DTT for 30 minutes at37° C., as well as these dialyzed samples, are then diluted to differentconcentrations in the latter HEPES buffer supplemented with 1 mM DTT.

[0114] Reaction mixtures include 5 μl recombinant SNAP-25 substrate (8.5μM final concentration) and either 20 μl reduced BoNT/A or recombinantwild-type L chain. All samples are incubated at 37° C. for 1 hour beforequenching the reactions with 25 μl aqueous 2% trifluoroacetic acid (TFA)and 5 mM EDTA (Foran et al., Biochemistry 33:15365(1994)). Aliquots ofeach sample are prepared for SDS-PAGE and Western blotting with thepolyclonal SNAP-25 antibody by adding SDS-PAGE sample buffer andboiling. Anti-SNAP-25 antibody reactivity is monitored using an ECLdetection system and quantified by densitometric scanning.

[0115] Western blotting results indicate clear differences between theproteolytic activities of the purified mutant L chain and either nativeor recombinant wild-type BoNT/A-L chain. Specifically, recombinantwild-type L chain cleaves the SNAP-25 substrate, though somewhat lessefficiently than the reduced BoNT/A native L chain that serves as thepositive control in the procedure. Thus, an enzymatically active form ofthe BoNT/A-L chain is produced by recombinant means and subsequentlyisolated. Moreover, substitution of a single amino acid in the L chainprotein abrogated the ability of the recombinant protein to degrade thesynaptic terminal protein.

[0116] As a preliminary test of the biological activity of the wild-typerecombinant BoNT/A-L chain, the ability of the MBP-L chain fusionprotein to diminish Ca²⁺-evoked catecholamine release fromdigitonin-permeabilized bovine adrenochromaffin cells is examined.Consistently, wild-type recombinant L chain fusion protein, eitherintact or cleaved with Factor X₂ to produce a mixture containing freeMBP and recombinant L chain, induced a dose-dependent inhibition ofCa²⁺-stimulated release equivalent to the inhibition caused by nativeBoNT/A.

EXAMPLE 8 Reconstitution of Native L Chain, Recombinant Wild-Type LChain with Purified H Chain

[0117] Native H and L chains are dissociated from BoNT/A (ListBiologicals Inc.; Campbell, USA) with 2 M urea, reduced with 100 mM DTTand then purified according to established chromatographic procedures(Kozaki et al., Japan J. Med. Sci. Biol. 34:61 (1981); Maisey et al.,Eur. J. Biochem. 177:683 (1988)). Purified H chain is combined with anequimolar amount of either native L chain or recombinant wild-type Lchain. Reconstitution is carried out by dialyzing the samples against abuffer consisting of 25 mM Tris (pH 8.0), 50 μM zinc acetate and 150 mMNaCl over 4 days at 4° C. Following dialysis, the association of therecombinant L chain and native H chain to form disulfide-linked 50 kDadichains is monitored by SDS-PAGE and quantified by densitometricscanning. The proportion of dichain molecules formed with therecombinant L chains is lower than that obtained when native L chain isemployed. Indeed, only about 30% of the recombinant wild-type or mutantL chain is reconstituted while >90% of the native L chain reassociatedwith the H chain. In spite of this lower efficiency of reconstitution,sufficient material incorporating the recombinant L chains is easilyproduced for use in subsequent functional studies.

[0118] While this invention has been described with respect to variousspecific examples and embodiments, it is to be understood that theinvention is not limited thereto and that it can be variously practicedwith the scope of the following claims. Other embodiments, versions, andmodifications within the scope of the present invention are possible.For example, from about 500 units to about 4,000 units of a botulinumtoxin type B can be used to treat an injured muscle according to thepresent disclosed invention.

We claim:
 1. A method for treating an injured muscle, the methodcomprising the step of local administration of a therapeuticallyeffective amount of a neurotoxin to an injured muscle, thereby treatingthe injured muscle.
 2. The method of claim 1 wherein the step of localadministration is by intramuscular injection.
 3. The method of claim 1wherein the neurotoxin substantially immobilizes the injured muscle. 4.The method of claim 1 wherein the neurotoxin is effective to immobilizethe injured muscle during phase 1 and phase 2 of a repair process of theinjured muscle.
 5. The method of claim 1 wherein the neurotoxin iseffective to immobilize the injured muscle during phase 1 of a repairprocess of the injured muscle.
 6. The method of claim 1 wherein theneurotoxin is a botulinum toxin type A, B, C₁, D, E, F or G.
 7. Themethod of claim 1 wherein the neurotoxin is a recombinantly madeneurotoxin.
 8. The method of claim 1 further comprising the step oftreating the injured muscle with physical therapy and/or surgery.
 9. Amethod for treating an injured muscle, the method comprising the step ofin vivo, local administration of a therapeutically effective amount of abotulinum toxin to an injured muscle, thereby treating the injuredmuscle.
 10. The method of claim 10, wherein the botulinum toxin isbotulinum toxin type A.
 11. A method for promoting healing of an injuredmuscle, the method comprising the step of in vivo, local administrationof a therapeutically effective amount of a botulinum toxin type A to aninjured muscle, thereby promoting the healing of the injured muscle. 12A method for treating pain associated with an injured muscle, the methodcomprising the step of in vivo, local administration of atherapeutically effective amount of a botulinum toxin to an injuredmuscle, thereby reducing the pain associated with an injured muscle. 13.The method of claim 12, wherein the botulinum toxin is botulinum toxintype A.